Exploring the Activity and Specificity of Gold Nanoparticle-Bound Trypsin by Capillary Electrophoresis with Laser-Induced Fluorescence Detection Yu-Fen Huang, Chih-Ching Huang, and Huan-Tsung Chang* Department of Chemistry, National Taiwan University, Taipei, Taiwan, Republic of China Received March 29, 2003. In Final Form: June 15, 2003 This paper describes the use of micellar electrokinetic chromatography (MEKC) and capillary zone electrophoresis (CZE) in conjunction with laser-induced fluorescence (LIF) detection for investigating the specificity of biocatalysis by trypsin when it is conjugated to gold nanoparticles (GNPs). In the presence of sodium dodecyl sulfate (SDS), adsorption of the tryptic fragments on GNP-trypsin and on the capillary wall is reduced. As a result, the sensitivity and resolution of electropherograms of the tryptic fragments from bovine serum albumin (BSA) is improved. MEKC-LIF measurements show clearly that the specificity of GNP-trypsin differs from that of free trypsin and that the tryptic digest of GNP-BSA is significantly different from the GNP-tryptic digest of BSA. We have used CZE-LIF to observe differences in the biocatalytic activity of trypsin and GNP-trypsin. Changes in the electropherograms provide information of the progress of digestion and indicate that the activity of GNP-trypsin is lower than that of free trypsin. The results of this study suggest that changes in the conformations and steric effects contribute to the loss of activity and changes in specificity of trypsin adsorbed on GNPs. Introduction Advances in nanotechnology allow the synthesis and fabrication of numerous novel nanoparticles (NPs) for different purposes, such as in electronics, as sensors, and as catalysts, because of the unique electronic, optical, and catalytic properties that result from their size. 1-3 These properties arise for the following reason: For a given mass of gold, the surface area increases with decreasing particle size. In addition, the selectivity and specificity of NPs toward substrates can be improved by controlling their sizes and shapes precisely and/or by modifying their surfaces. Functional gold nanoparticles (GNPs) that are conju- gated with biomolecules such as DNA, enzymes, and antibodies have been made and demonstrated in a variety of fields, including biosensors, separation science, and catalysis. 4-11 Recently, GNPs conjugated with enzymes, such as pepsin and fungal protease, have been synthesized and tested, with results showing that biocatalytic activity and stability in aqueous solution is retained. 10,11 The conjugation is believed to occur mainly through interac- tions of the gold surface with thiol groups of cysteine residues and amino groups of lysine residues of the enzymes; the stability of bioconjugated GNPs is attributed to steric and Coulombic repulsions. To retain their biocatalytic activities, the three-dimensional structures of enzymes must still exist when they are bound to GNPs. This situation can be achieved by carefully modifying the GNP surface with suitable capping agents, such as citrate, and/or with spacers, such as poly(ethylene glycol). 12 In addition, the pH and ionic strength of the aqueous solution are important factors that affect catalytic behavior. For example, the biological activity (selectivity and digestion efficiency) of pepsin-bioconjugated GNPs remains almost the same as it is in the bulk solution at pH 3.0 when glycine is used as the capping agent. 10 Numerous spectroscopic tools, such as UV-vis absorp- tion, fluorescence, Raman scattering, and IR spectroscopy, are commonly used to explore the stability and activity of bioconjugated GNPs. For example, the shifts in the so-called amide I and II bands in IR spectra of enzymes, and the π-π* band in fluorescence spectra that is due to the tryptophan residues, have been utilized to investigate adsorption, while acid-soluble products (tryptophan and tyrosine residues) have been used to determine the biocatalytic ability of bound enzymes by measuring the UV absorbance of aromatic units at 280 nm. 10,11,13 These techniques, however, do not provide direct information on the digestion products of NP-bound enzymes, which is information that is essential when investigating the biocatalytic activity and specificity of enzymes. Capillary electrophoresis (CE) is a fast and efficient separation technique that can be used for the analysis of tryptic digests. 14 It is a long-standing interest in our research group to study protein separation by CE in conjunction with laser-induced fluorescence (LIF) detec- * Corresponding author: Tel/fax 011-886-2-2362-1963; e-mail changht@ntu.edu.tw. 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